Phosphatidylserine-dependent Phagocytosis
Phosphatidylserine is a negatively charged phospholipid which is located in the inner layer of the plasma membrane in all cells. Occasionally, however, a phosphatidylserine molecule translocates to the outer layer of the plasma membrane. In living healthy cells, phosphatidylserine which has reached the outer layer is immediately transported back enzymatically to the inner layer of the plasma membrane. In contrast, the phosphatidylserine remains in the outer cell membrane layer in aged and in Plasmodium falciparum-infected erythrocytes, in sickle cells, post-inflammatory granulocytes and in apoptotic cells. If a certain degree of phosphatidylserine exposure is reached, phagocytes bind to these cells, which still maintain the integrity of their plasma membranes, via the phosphatidylserine receptor. If the phosphatidylserine density reaches a certain threshold value, the cells which are committed to die are very rapidly phagocytozed. In this process, no release of the cell contents to the surrounding tissue and therefore no activation of the immune system occurs. For this reason, this phagocytosis pathway, which depends on the recognition of phosphatidylserine on the surface of a dying cell, is called non-inflammatory.
The Role of Phosphatidylserine-dependent Phagocytosis in Malaria-infections
In the course of physiological tissue turn-over, when cells which have grown old are removed, e.g. erythrocytes and apoptotic cells, such as post-inflammatory granulocytes, a specific immunosuppression is essential, since in these cases a pro-inflammatory phagocytosis would result in autoimmune phenomena. The non-inflammatory phagocytosis of Plasmodium falciparum-infected erythrocytes, however, is responsible, inter alia, for the extremely poor immune response and difficulties in the immunization against malaria. No measure described to date or prophylaxis against malaria considers the circumstance in which Plasmodium falciparum-infected erythrocytes are taken up into phagocytes by phosphatidylserine-dependent phagocytosis. Medicaments which affect this phagocytosis pathway are presently unknown.
The Role of Phosphatidylserine-dependent Phagocytosis in Viral Infections
The role of the phosphatidylserine-dependent phagocytosis pathway is similar in viral infections. Viruses which are taken up into phagocytes through the phagocytosis of virus-infected apoptotic cells can thus escape immunosurveillance. The uptake of HIV in monocytes, for example, which takes place without triggering of the “respiratory burst”, is responsible for the penetration of the HIV into the long-lived monocyte pool, which is early and unnoticed by the immune system. This infection of the monocytes/macrophages, which is presently not understood, is held causally responsible for the persistence of HIV and thus for the formation of the AIDS syndrome. Although the route of infection of monocytes/macrophages with HIV is presently still not clearly identified in molecular terms, an involvement of phosphatidylserine and the phosphatidylserine receptor is probable because of the non-inflammatory phagocytosis. It was for example possible to show, that retrovirus genomes from apoptotic cell debris can be taken up into cells and cause an infection of these cells. Since HIV can survive for a very long time in monocytes, and is possibly spontaneously released even years after the infection, the human immune system cannot completely eliminate the HIV from the body. Since the HIV damages the immune system somewhat on each release by destroying the CD 4-positive cells, the full degree of the AIDS syndrome can thus take several years to develop. Similar problems also exist in the elimination of other viruses persisting or replicating in phagocytes.
Other retroviruses and particularly the subgroup of the lentiviruses can especially be mentioned here. Some of these viruses (e.g. EIAV, Maedi Visna Virus, CAEV) persist in the phagocytes of hoofed animals and lead to autoimmune diseases. No previously described measure or prophylaxis against HIV infection or infection with other viruses surviving in phagocytes considers the circumstance in which apoptotic cells can be phagocytozed via the phosphatidylserine-dependent pathway. Medicaments which block or modify this phagocytosis pathway are presently unknown.
The Role of Phosphatidylserine-dependent Phagocytosis in Sickle Cell Anemia
The situation is different in patients with sickle cell anemia. Owing to the continuous and extremely rapid phagocytosis of autologous, genetically modified erythrocytes, anemia occurs in these patients, which can lead to death in severe cases if untreated. Here, the fact that the phosphatidylserine mediated phagocytosis proceeds in a non-inflammatory manner is less prominent than the fact that phosphatidylserine-exposing cells are eliminated in an extremely rapid and efficient way. Since there are no medicaments which block or modify this phagocytosis pathway, sickle cell anemia is presently treated with repeated blood transfusions.
The Role of Phosphatidylserine-dependent Phagocytosis in Erythrocyte Stability
A problem similar to that in sickle cell anemia also occurs in the storage of erythrocytes for transfusion. Even under blood bank conditions, an increasing number of erythrocytes exposes phosphatidylserine on their surface during storage. After the transfusion, these erythrocytes are very rapidly cleared by phagocytes and thus are lost. Moreover, the transfusion of a substantial amount of aged erythrocytes exposing phosphatidylderine on their surfaces can be stressful to the recipient's organism. Since there are presently no medicaments or additives to conserved blood which prevent this phagocytosis, the storage of erythrocytes is strictly limited in terms of time.
The Role of Phosphatidylserine-dependent Phagocytosis in Cancer Therapy
Tumor vaccines prepared of autologous apoptotic cancer cells, after injection are usually rapidly eliminated by macrophages via anti-inflammatory phagocytosis and therefore do not result in an efficient sensitization of the immune system to the tumor.
In the preparation of tumor vaccines, the tumor cells returned to the bodies of patients or experimental animals are irradiated in order to prevent the formation of metastases. Since under these circumstances apoptosis is induced in the tumor cells and these are then eliminated in a non-inflammatory manner via the phosphatidylserine-dependent phagocytosis pathway, only a relatively weak immune response usually occurs to the respective tumor. Since at present no substances are known which block or modify the phosphatidylserine-dependent phagocytosis pathway, classical immunization routes and adjuvants are currently used in order to increase the immune response to tumor cells.
Cancer vaccines pursue the strategy of a specific activation of the immune system to achieve the recognition and elimination of the tumor. One possibility is the use of whole tumor cells as vaccines since they display cancer-associated antigens as the immunological key to the destruction of the tumor they were derived from. An obstacle of these vaccinations is the weak immunogenicity of cancer cells alone, which could be overcome by the additional use of immunostimulatory or response modifying molecules. Annexin V is a monomeric protein ligand of anionic phospholipids and exhibits high affinity to membrane bound phosphatidylserine, which is translocated from the inner to the outer cell membrane layer in apoptotic cells. Apoptotic tumor cells do express phosphatidylserine and consequently might maintain an anti-inflammatory and non-immunogenic environment. Apoptotic tumor cells coated with chicken annexin V lack the phosphatidylserine signal on their surface which reduces the interaction with its receptor. In this case, phagocytosis occurs via different receptors, inducing macrophages to secrete pro-inflammatory mediators and dendritic cells to migrate and maturate, thus achieving a specific immune response against the tumor.
Immunological Background
Regulation of the cell number is a key process to normal development and hemostasis in the healthy adult. Organisms keep the correct number of cells by a genetically controlled and well-regulated process of programmed cell death called apoptosis (Kerr, J. F. R., Wyllie, A. H., Currie, A. R. 1972. Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br. J. Cancer 26, 239-57). Typical apoptotic features of a cell are nuclear condensation, cell shrinkage (in opposite to the swollen appearance of necrotic cells), membrane blebbing and protein and DNA fragmentation.
A crucial part of apoptosis is the removal of the intact dying cell from the tissue before it causes inflammatory responses. This occurs through phagocytosis by macrophages. Phospholipids are asymmetrically distributed between the inner and outer layer of the plasma membrane with phosphatidylcholine and sphingomyelin exposed on the external layer of the membrane, and phosphatidylserine, phosphatidic acid and phosphatidylethanolamine predominantly observed on the inner surface facing the cytosol. In cells undergoing apoptosis, as well as after platelet activation or endothelial cell injury, phosphatidylserine is translocated to the outer layer of the membrane and is one of the “eat-me” ligands present on the cell surface during apoptosis (Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L., Henson, P. M. 1992. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207-2216). Once committed to die, the cell exposes phosphatidylserine at its surface within minutes while maintaining the integrity of the plasma membrane. Macrophages recognize phosphatidylserine via a specific receptor and thereby activate intracellular pathways which orchestrate uptake of the apoptotic cell.
Phosphatidylserine exposed at the cell surface exhibits pro-coagulant activities. Annexin V, originally discovered as an anticoagulant with an antithrombotic activity in vivo, binds with high affinity to phosphatidylserine on apoptotic cells and thereby impairs the pro-coagulant activities of the dying cell (Reutelingsperger, C. P. M. and van Heerde, W. L. 1997. Annexin V, the regulator of phosphatidylserine-catalyzed inflammation and coagulation during apoptosis. Cell. Mol. Life Sci. 53, 527-32). Fluorescently labeled annexin V is routinely used for the detection of apoptotic cells in flow cytometric assays (Van Engeland, M., Nieland, L. J. W., Ramaekers, F. C. S., Schutte, B., Reutelingsperger, C. P. M. 1998. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31, 1-9).
The immune system has to contend with the consequences of two types of cell death, necrosis and apoptosis. Apoptosis, as described above, is the physiological form for the non-inflammatory removal of intact dying cells during normal tissue turn over. Unremoved apoptotic cells further proceed to the stage of secondary necrosis and are identically handled. Primary necrosis, in contrast, is a pathological event resulting in cell lysis and consequently in the induction of inflammation.
Two different types of antigen presenting cells (APC), macrophages and dendritic cells (DC), are involved in the clearance of dying cells: Macrophages degrade and process antigens contained within apoptotic cells, but they fail to induce antigen-specific cytotoxic T lymphocytes (CTL) when injected in vivo. After phagocytosis of apoptotic cells macrophages modulate the immune response by the release of immunosuppressive factors and the failure to present antigen, whereas exposure to primary necrotic cells leads to activation of macrophages towards inflammation. The response against secondary necrotic cells comprises features of the response against apoptotic cells as well as features of the response against necrotic cells.
Optimal cross-presentation of antigens acquired from dying cells by DC requires two steps: (i) phagocytosis of dying cells in the immature DC state and (ii) receipt of an appropriate maturation signal. Immature DC are located in the body's periphery, capture antigen and thus receive a signal to leave the tissue and migrate to the regional lymph node. A maturation signal can be provided e.g. by necrotic cell fragments and leads to a 5-10 fold improved antigen presentation via upregulation of MHC (major histocompatibility complex) and costimulatory molecules and the capacity to induce antigen specific CD4+ and CD8+ T cells. In contrast, phagocytosis of apoptotic cells in early stages of apoptosis fails to induce full maturation and may lead to the induction of tolerance to self and considerably low immunogenicity. (Sauter, B., Albert, M. L., Francisco, L., Larsson, M., Somersan, S., Bhardwaj, N. 2000. Consequences of cell death: Exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells. J. Exp. Med. 191, 423-433).
Autologous apoptotic tumor cells are scavenged and degraded by macrophages, thereby preventing accumulation of high amounts of autoantigens in areas of cell death. Under these conditions macrophages actively suppress autoimmune responses through production of anti-inflammatory cytokines like transforming growth factor β (TGF-β), interleukin (IL)-10, platelet activating factor (PAF), and prostaglandin E2 (PGE2) and through inhibition of pro-inflammatory cytokines like IL-1β, tumor necrosis factor (TNF)-α, granulocyte macrophage-colony stimulating factor (GM-CSF), IL-12 and IL-8 (Voll, R. E., Hermann, M., Roth, E. A., Stach, C., Kalden, J. R. 1997. Immunosuppressive effects of apoptotic cells. Nature 390, 350-351; Fadok, V. A., Bratton, D. L., Konowal, A., Freed, P. W., Westcott, J. Y., Henson, P. M. 1998. Macrophages that have ingested apoptotic cells in vitro inhibit pro-inflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-β, PGE2, and PAF. J. Clin. Invest. 101, 890-898). Immature dendritic cells fail to receive an adequate full maturation signal after phagocytosis of apoptotic tumor cells. The overall result is low immunogenicity of apoptotic tumor cells. Tumors most probably undergo some level of cell turn over through the cell death mechanism of apoptosis thereby circumventing an effective immune response.
It is believed that the phosphatidylserine receptor (PSR) serves as a crucial switch that controls the development of inflammation and/or the initiation of the adaptive immune response. (Henson, P. M., Bratton, D. L., Fadok, V. A. 2001. The phosphatidylserine receptor: a crucial molecular switch? Nature Rev. Mol. Cell Bio. 2, 627-633). This theory is supported by the fact that macrophages and DC are obviously able to distinguish, whether a cell is going to die by apoptosis or necrosis.
For cases in which sufficient ligation of the PSR occurs, e.g. by an apoptotic cell which expresses phosphatidylserine externally, suppression may dominate: downregulation of pro-inflammatory cytokines through the release of TGF-β by macrophages is induced and DC-maturation inhibited.
Uptake of apoptotic and remarkably also secondary necrotic cells does not result in DC maturation and antigen presentation, but primary necrotic cells, particularly tumor cells, can activate the response. In this case, when ligation of the PSR is insufficient, the pro-inflammatory and immune-stimulatory effects take over: secretion of pro-inflammatory cytokines and maturation of DC are induced. Thus, APC are able to distinguish two types of cell death, with primary necrosis providing a critical signal that will promote the initiation of immunity.
There are two possible mechanisms to overcome the dominant anti-inflammatory effect of PSR ligation. The susceptibility of the PSR to protease cleavage leads to the assumption that proteases e.g. released by primary necrotic cells would remove the PSR receptor from the cell surface resulting in transiently unprotected cells that could be triggered to produce pro-inflammatory mediators and/or to fully mature towards an antigen-presenting and immune response stimulating dendritic cell (Fadok, V. A., Bratton, D. L., Guthrie, L. A., Henson, P. M. 2001. Differential effects of apoptotic vs. lysed cells on macrophage production of cytokines: role of proteases. J. Immunol. 166, 6847-6854).
Another possible mechanism is the assumption that proteins known to bind phosphatidylserine with high affinity, such as annexins, will be able to block the exposed phosphatidylserine to reduce its interaction with the PSR. Annexin V coating the surface of an apoptotic cell and thereby shielding the PSR most probably reduces the interaction of phosphatidylserine with its receptor. Several other cell surface or bridging molecules interacting with phosphatidylserine might also be impaired. The overall result would be the loss of the dominating PSR function, the stimulation of macrophages to create a pro-inflammatory environment and a signal for the full maturation of dendritic cells, their migration to lymph nodes, and immune stimulatory antigen presentation, resulting in the stimulation of a specific T cell response.
According to the prior art, annexins are additionally known. Annexin V is a member of a ubiquitously occuring family of annexin proteins which share structural and functional features (Mollenhauer, J. 1997. Multi-author review. Annexins: what are they good for? Cell Mol. Life Sci. 53, 506-556). The common property of annexins is the reversible calcium-dependent binding to anionic phospholipid membranes. Although this property may be due to highly conserved sequences, the existence of at least 13 different annexins in mammalian species suggests that they have specific and diverse biological functions. These functions relate to membrane associated processes, and recent data show their function in the regulation of thrombosis, hemostasis and apoptosis.
Specificity and diversity of annexins may be provided by their N-terminal domains (which are less conserved among different members of the annexin family), but may also be the consequence of interactions of annexins either with other members of this protein family or other cellular partners. Typically, annexins have molecular weights ranging between 30 and 40 kD (only annexin VI is exceptional with regard to its molecular weight of 66 kD).
The annexins also participate in intracellular membrane trafficking during exo- and endocytosis, phagosome formation, and lipid raft clustering (reviewed in Reutelingsperger, C. P. M. 2001. Annexins: key regulators of haemostasis, thrombosis, and apoptosis Thromb. Haemost. 86, 413-419). They inhibit cytosolic phospholipases and protein kinases. Annexin V also possesses a voltage-dependent Ca2+ ion channel activity (Voges, D., Berendes, R., Demange, P. et al. 1995. Structure and function of the ion channel model system annexin V. Adv. Enzymol. Relat. Areas Mol. Biol. 71, 209-239). This kind of activity needs penetration of annexin V into the hydrophobic core of the lipid bilayer, which was recently shown under mild acidic conditions (pH 5-6) (Isas, J. M., Cartailler, J. P., Sokolow, Y., Patel, D. R., Langen, R., Luecke, H., Hall, J. E., Haigler, H. T. 2000. Annexins V and XII insert into bilayers at middly acidic pH and form ion channels. Biochemistry 39, 3015-22).
While many annexin functions are intracellular, others occur outside the cell. Annexin V was found in extracellular fluids like cerebrospinal fluid and blood plasma. The pathway of its externalisation is not fully understood. Once present in the extracellular space, the annexins have been shown to function as receptors for many polypeptide ligands and exhibit a variety of extracellular activities: annexin V binds with high affinity to phospholipid membranes of platelets, inhibits lipid-dependent reactions of the blood coagulation and intravascular thrombus formation, binds to collagens and exhibits lectin activity, i.e. binds to carbohydrate moieties of glycoproteins (Seaton, B. A., Dedman, J. R. 1998. Annexins. Bio. Metals 11, 399-404; Turnay, J., Pfannmüller, E., Lizarbe, M. A., Bertling, W., von der Mark, K. 1995. Collagen binding activity of recombinant and N-terminally modified annexin V (anchorinCII) J. Cell. Biochem. 58, 208-220; Mollenhauer, J. 1997. Multi-author review. Annexins: what are they good for? Cell Mol. Life Sci. 53, 506-556).
Recently, a new class of diseases called the “annexinopathies” was postulated, characterized by an aberrant expression of annexin II or V (Rand, J. H. 1999. “Annexinopathies”—A new class of diseases. N. Engl. J. Med. 340, 1035-36). These diseases strongly indicate that these annexins exhibit a role in the physiological control of blood coagulation.
The medicaments and procedures known according to the prior art, in particular the adjuvants employed today, stimulate the immune system non-specifically. To date, no agent is yet described which prevents or modifies the phagocytosis of phosphatidylserine-exposing cells, thus leading to a specific immunostimulation. The disadvantages which occur in non-inflammatory clearing of whole cell vaccines and virusinfected cells are particularly to be emphasized. On the one hand, phosphatidylserine-dependent phagocytosis contributes to the ineffectivity of vaccines through their breakdown, on the other hand to virus persistence.
Since, in sickle cell anemia, even young erythrocytes expose phosphatidylserine on their surface, they are removed by the endogenous phagocytes. This contributes disadvantageously to the anemia of the patients.
Even for the storage of blood and erythrocytes, no medicaments or additives are known which prevent the breakdown of the donor erythrocytes by the recipient's phagocytes after transfusion. This presently leads to a relatively short shelf life of conserved blood and erythrocyte concentrates and to a marked loss of activity in preserves stored for a long time period.
The object of the present invention is to eliminate the disadvantages according to the prior art. In particular, a medicament or the use of an active compound which brings about an increase in the immunity to viruses, tumors, bacteria and parasites will be specified.